Abstract Current treatments of Type 2 diabetic patients for hyperglycemia result in constitutive and non-regulated insulin delivery, and this failure to mimic endogenous insulin secretion leads to hypoglycemia problems and eventual beta cell failure. Thus the long term goal here is to decipher the mechanisms used by pancreatic islet beta cells to meter insulin release, and then devise ways to recapitulate this metering mechanism pharmacologically in the patient. Thousands of insulin granules exist behind a filamentous actin (F-actin) barrier in the beta cell and F-actin remodeling is known to mobilize granules to the t-SNARE proteins at the cell surface, yet the mechanisms involved in remodeling and granule mobilization are largely unknown and untested. Published and preliminary work presented here suggests that the key to actin remodeling lies in the glucose-specific activation of the small Rho family GTPase protein Cdc42, and that Cdc42 is essential for second-phase insulin release from islets. New data also reveal that the Cdc42 guanine dissociation inhibitor (GDI) is required to keep Cdc42 inactive, and its mutation/depletion leads to inappropriate constitutive insulin secretion. The Cdc42-GDI complex binds directly to the v-SNARE on the insulin granules, and disruption of this binding attenuates glucose but not KCl-stimulated insulin secretion. Moreover, this glucose-specific Cdc42 activation is coupled to interactions between t-SNARE proteins and F-actin. Thus, the objective of this application is to delineate the physiologic, cellular and molecular mechanisms by which glucose activates Cdc42 to promote second-phase insulin release, and to determine how Cdc42 controls second phase through Cdc42-v-SNARE interactions and by impacting F-actin-t-SNARE associations to regulate insulin granule targeting and actin reorganization. The central hypothesis for the proposed research is that that Cdc42 becomes activated specifically in response to glucose to coordinate the second phase of insulin release by selectively remodeling F-actin to mobilize and target granules towards SNARE sites at the plasma membrane for exocytosis. This will be tested in three Specific Aims: 1) Elucidate how Cdc42 activation is regulated by glucose in pancreatic beta cells; 2) Identify how Cdc42-v-SNARE interactions regulate insulin exocytosis; 3) Determine the functional role of actin-t-SNARE interactions in Cdc42-mediated actin remodeling and the regulation of insulin exocytosis. Studies will be accomplished using siRNA-mediated knockdown in islet beta cells with 'rescue' strategies, and by corroborating quantitation of biphasic insulin release (islet perifusion) with visualization of spatial changes in Cdc42 interactions using microscopy and biochemical subcellular fractionation analyses. Gaining knowledge of how Cdc42 functions in second-phase secretion will mark progress towards the long-term goal of modulating actin remodeling in the beta cell to recapitulate regulated insulin secretion and prevent beta cell failure. ProjectNarrative Althoughdiabeticpatientsusesulfonylureasorinsulininjectiontosurvive,thesetreatmentsfailto simulatethecarefullymeteredreleaseofinsulinthatwouldotherwisecomefromtheirownpancreaticislet cells.Ourpreliminarydatahaverevealedthatthemeteredreleaseofinsuliniscontrolledbyaproteinnamed Cdc42,andfurtherinvestigationofthisprotein'sfunctionininsulinsecretionwillhopefullyleadtodiscovery ofnewtherapeuticstrategiesforinsulindeliverythatbettersimulatenaturalinsulinreleasefromahealthy humanpancreastoimprovethelivelihoodofpeoplewithdiabetes.